Comparative genomics is a powerful tool for uncovering patterns of mutations within genes targeted in human health concerns. To examine structure and function, we sequence targeted genes across multiple species to identify the pattern and rate of substitutions. This report summarizes our investigation of four genes linked with varying extent to reproductive fitness located on the Y-chromosome. A unique feature of the Y-chromosome is that genes known to be important in male fertility are located within the non-recombining region of the Y (NRY) chromosome. During cell division the NRY does not align with the X-chromosome and prevents conventional recombination. As a result, the NRY was thought originally to be a "functional wasteland" and these genes were expected to either become specialized for male function, or gradually degenerate from the accumulation of deleterious mutations. Yet, research has indicated that functional NRY genes include "house-keeping" genes that are expressed ubiquitously. Understanding how genes maintain function in the absence of conventional recombination begins with determining the pattern of changes that accumulate within the sequence over time. To obtain this information, we employ comparative phylogenetic analyses of sequence data across species within a reference taxonomic group. We use the cat family Felidae as the reference phylogeny based on concordant results from multiple genetic studies that resolve the pattern of evolution of the 37 extant species from a common ancestor 12 MYA. We sequenced NRY genes across all species of cat, and estimated patterns of mutations within these genes. In 2004, we have completed and published an analysis of intron regions (3604 bp) within three Y-chromosome genes: zinc-finger protein on the Y (ZFY), select mouse cDNA on Y (SMCY) and ubiquitin activating enzyme E1 on Y (UBE1Y) in Felidae. Currently, we are comparing these intronic data with sequences from the protein-coding regions of these genes to examine relative patterns of changes. We are investigating the relative differences in rates of change between the sex chromosomes (X and Y) as well as autosomes. Further, we are completing our analysis of (sex-determining region of the Y)(SRY): a single copy gene exclusive to the Y-chromosome that is the trigger for male sex determination during embryonic development. We compare and contrast patterns of evolution within SRY (705 bp) with sequence from about 2 kb of noncoding DNA from the adjacent genomic flanks. Our results indicate SRY is under strong purifying selection to maintain function. At present, using the recently published human Y-chromosome sequence as a guide, we are characterizing gene content and order within the domestic cat Y-chromosome. Our efforts are focused on the development and implementation of multiple methods to efficiently identify genes and map their position on the cat Y-chromosome. By comparing cat with mouse and human, new insights onto the evolution, structure and function of genes in the NRY are possible.